Therapy for cancer has advanced significantly. Many proliferative disorders can now be effectively treated by administering therapeutic agents that include natural products, derivatives of natural products and synthetic compounds. Therapy for proliferative disorders, particularly cancer chemotherapy, may comprise administration of a combination of agents.
I. Myelodysplastic Syndromes and Myeloid Leukemias
Myelodysplastic syndrome (MDS), derived from a multipotent hematopoietic stem cell, is characterized clinically by a hyperproliferative bone marrow, reflective of ineffective hematopoiesis, and is accompanied by one or more peripheral blood cytopenias. Bone marrow failure results, leading to death from bleeding and infection in the majority, while transformation to acute leukemia occurs in up to 40% of patients. Because of the high rate of transformation to acute leukemia, myelodysplastic syndrome has also been termed “preleukaemia”. Clinical aspects of the disease are reviewed by L. R. Silverman in Cancer Medicine, Ed. David W. Kufe et al., 6th Edition, B. C. Decker, 2003, the entire disclosure of which is incorporated herein by reference. See also: P. L. Greenberg, N. S. Young, and N. Gattermann, “Myelodysplastic Syndromes”, Hematology, 2002, 136-61.
Estimates of the incidence of myelodysplastic syndrome range from 1 case per 100,000 per year to a frequency approximately equal to or greater than that of acute myeloid leukemia (AML) or approximately 14,000 new cases per year in the United States. The incidence appears to be increasing, which is probably due to a number of factors including greater awareness, greater diagnostic precision and the aging of the population.
The French-American-British (FAB) Study Group has recognized five categories of myelodysplastic syndrome based on morphologic characteristics and the percentage of blasts in the bone marrow and peripheral blood:                Refractory anemia (RA): Patients suffer from an anemia that is resistant or refractory to treatment with iron and/or vitamins, and there are less than 5% blasts in the marrow. There may be mild to moderate lowering of WBC and platelets as well.        Refractory anemia with ringed sideroblasts (RARS): Patients have a refractory anemia, and in addition, abnormal precursors of red cells containing iron deposits in the form of a ring around the nucleus called ringed sideroblasts make up more than 15% of the marrow cells. Blasts constitute less than 5% of the marrow.        Refractory anemia with excess blasts (RAEB): In this category, patients with refractory anemia also have either 1-5% circulating blasts in the peripheral blood or 5-20% blasts in their marrow.        Refractory anemia with excess blasts in transformation (RAEB-t): If the percentage of circulating blasts exceeds 5%, or there are 20-30% blasts in the bone marrow, the patients are considered as transforming towards acute leukemia.        Chronic myelomonocytic leukemia (CMML): While the bone marrow looks more or less similar to the other types of myelodysplastic syndrome, there is an increase in monocyte cells in both blood and marrow, and the total WBC count may also be increased. The blasts are between 5-20% in the bone marrow.        
A classification system has also been developed by the World Health Organization, which can be related to the FAB classification as shown in Table 1.
TABLE 1Classifications of Myelodysplastic SyndromeFAB ClassificationWHO ClassificationRefractory anemia (RA)Refractory anemia (unilineage)5q-syndromeRefractory cytopenia with multilineagedysplasia (RCMD)Refractory anemia withRefractory anemia with ringed ringed sideroblasts (RARS)sideroblasts (unilineage) 5q-syndromeRefractory cytopenia with multilineagedysplasia and ringed sideroblasts(RCMD-RS)Refractory anemia withRefractory anemia with excess blasts Iexcess blasts (RAEB)(RAEB-I)Refractory anemia with excess blasts II(RAEB-II)Refractory anemia with ex- (Classified as acute myeloid leukemia)cess blasts in transformation (RAEB-t)Chronic myelomonocytic Chronic myelomonocytic leukemia (CMML)leukemia (CMML)Unclassifiable myelodysplastic syndrome
The cellular elements of blood originate from the pluripotent hematopoietic stem cell. Stem cells have extensive regenerative and differentiating capacity and generate lymphoid and myeloid precursors, which then produce lymphocytes, neutrophils, eosinophils, basophils, erythrocytes, and platelets. In myelodysplastic syndrome, a dysregulation in the differentiation process appears to occur. Mortality in myelodysplastic syndrome is related to bleeding, recurrent infection, and leukemic transformation. In the absence of treatment, myelodysplastic syndrome can be a rapidly fatal disease, with or without the transformation to acute myeloid leukemia. An estimated 20-40% of adults with myelodysplastic syndrome develop leukemia, and 30-40% of myelodysplastic syndrome patients succumb to infection, bleeding, or both.
A prognostic scoring system, the International Prognostic Scoring System (IPSS), has been developed for patients with myelodysplastic syndrome. The IPSS is a consensus prognostic scoring system based on cytogenetic, morphological, and clinical data from seven large risk-based studies that had each generated prognostic systems. P. Greenberg, et al., “International Scoring System for Evaluating Prognosis in Myelodysplastic Syndromes”, Blood, 1997, 89(6) 2079-88. Compared with prior risk-based classifications, the IPSS provides an improved method for evaluating prognosis in MDS. Based on univariate analysis it was found that the major variables having an impact on disease outcome for evolution to acute myeloid leukemia were cytogenetic abnormalities, the percentage of bone marrow myeloblasts, and the number of cytopenias. Factors for survival, in addition to the above variables, also included age and gender.
The cytogenetic subgroups of outcome were classified as follows:
“good” outcomes were normal, ìY alone, del(5q) alone, del(20q) alone;
“poor” outcomes were complex (ie, δ3 abnormalities) or chromosome 7 anomalies;
“intermediate” outcomes were other abnormalities.
Multivariate analysis combined these cytogenetic subgroups with the percentage of bone marrow blasts and the number of cytopenias to generate a prognostic model. Weighting these variables by their statistical power separated patients into distinctive subgroups of risk for 25% evolution to acute myeloid leukemia:
low, 9.4 years;
intermediate-1 (INT-1), 3.3 years;
intermediate-2 (INT-2), 1.1 years; and
high, 0.2 year
These same features also separated patients into similar distinctive risk groups for median survival:
low, 5.7 years;
INT-1, 3.5 years;
INT-2, 1.2 years; and
high, 0.4 year.
The IPSS scoring system for myelodysysplastic syndrome is summarized in Table 2. The correlation between the IPSS score and median survival and progression to acute myeloid leukemia is summarized in Table 3, which also shows that stratification of IPSS scores for age further improves analysis of survival.
TABLE 2International Prognostic Scoring System (IPSS) for MyelodysplasticSyndrome Survival and Acute Myeloid Leukemia Progression.Score ValuePrognostic Variable00.51.01.52.0Bone Marrow Blasts (%)<55-10—11-2021-30Karyotype*GoodIntermediatePoor——Cytopenias0/12/3———*Good: normal, -y, del(5q), del(20q); Poor: complex (≧3 abnormalities) or chromosome 7 abnormalities; Intermediate: all other abnormalitiesScores for risk groups are as follows: Low, 0; INT-1, 0.5-1.0; INT-2, 1.5-2.0; High ≧ 2.5.
TABLE 3Age-Related Survival and Acute Myeloid Leukemia Evolution ofMyelodysplastic Patients Within the IPSS SubgroupsNo. ofIPSS ClassificationPatientsLowINT-1INT-2HighMedian Survival (yr)Total no.816 267 (33%)314 (38%)176 (22%)59 (7%)of patientsMedian (yr) 5.73.51.20.4Age≦60205 (25%)11.85.21.80.3>60611 (75%) 4.82.71.10.5≦70445 (54%) 9.04.41.30.4>70371 (46%) 3.92.41.20.425% Acute Myeloid Leukemia Evolution (yr)Total no.759 235 (31%)295 (39%)171 (22%)58 (8%)of patientsMedian (yr) 9.43.31.10.2Age≦60187 (25%)>9.4 (NR)6.90.70.2>60572 (75%) 9.42.71.30.2≦70414 (55%)>9.4 (NR)5.51.00.2>70345 (45%)>5.3 (NR)2.21.40.4NR: Not reached (i.e. fewer than 25% of the patient group progressed to AML)
Acute myeloid leukemia is the most common variant of acute leukemia occurring in adults, comprising approximately 80-85% of cases of acute leukemia diagnosed in individuals greater than 20 years of age. The heterogeneous group of acute leukemic disorders of myeloid hematopoietic cells has been called a variety of names including acute myelogenous leukemia, acute myelocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute granulocytic leukemia, and acute nonlymphocytic leukemia. The myeloid character of the malignant blasts can be determined by detection of characteristic morphologic and immunologic findings. A National Cancer Institute-sponsored workshop has suggested that the term acute myeloid leukemia (acute myeloid leukemia) is preferred. Clinical aspects of the disease are reviewed by C. A. Schiffer and R. M. Stone in Cancer Medicine, Ed. David W. Kufe et al., 6th Edition, B. C. Decker, 2003, the entire disclosure of which is incorporated herein by reference.
This French, American, and British (FAB) classification has been developed to diagnose and classify acute myeloid leukemia. The diagnosis of acute myeloid leukemia requires that myeloblasts constitute 30% (or 20% based on a recent World Health Organization (WHO) classification system) or more of bone marrow cells or circulating white blood cells. The hematologic properties of the disease, defines the various subtypes described below. The FAB nomenclature (M1 through M7) classifies the subtypes of acute myeloid leukemia according to the normal marrow elements that the blasts most closely resemble. Table 4 includes both the FAB classifications as well as additional classes recognized by the WHO.
TABLE 4Classifications of Acute Myeloid LeukemiaAcute myeloid leukemia, minimally differentiated (MO)Acute myeloid leukemia without maturation (M1)Acute myeloid leukemia with maturation (M2)Acute myeloid leukemia with maturation with t(8; 21)Acute promyelocytic leukemia (M3)Hypergranular typeMicrogranular typeAcute myelomonocytic leukemia (M4)Acute myelomonocytic leukemia with increased marrow eosinophils(M4EO)Acute Monocytic Leukemia (M5)Acute monoblastic leukemia (M5a)Acute monocytic leukemia with maturation (M5b)ErythroleukemiaErythroid/myeloid) (M6a)Pure erythroid malignancy (M6b)Acute megakaryoblastic leukemia (M7)Acute megakaryoblastic leukemia associated with t(1; 22)Acute basophilic leukemiaAcute myelofibrosis (acute myelodysplasia with myelofibrosis)Acute leukemia and transient myeloproliferative disorder in Down'sSyndromeHypocellular acute myeloid leukemiaMyeloid sarcoma
Although there have been gradual improvements in the complete remission (CR) rates worldwide in acute myeloid leukemia patients, this has not translated into improved outcomes, particularly for older patients. Reduced morbidity and mortality can be attributed to more widespread availability of sophisticated supportive care rather than new therapies. Relatively few changes in therapy have been made since the introduction of combined therapy with daunorubicin and cytosine arabinoside. Attempts to find new therapies have been disappointing. Combinations with alternative anthracyclines or other agents such as rubidizone, aclacinomycin, amsacrine, mitoxantrone, and idarubicin have been used in several trials, but none of these studies demonstrated a survival or disease-free survival advantage with these different agents. Overall, complete remission rates for treated patients are about 50-75%. However, in acute myeloid leukemia patients over 60 years of age the complete remission rate is only about 50%, with failures divided equally between drug resistant leukemia and deaths occurring during marrow aplasia as a consequence of reduced end organ tolerance. Complete remission can be achieved in only approximately 20%-30% of patients whose leukemia followed treatment for another cancer. Even if remission is achieved, however, because some leukemia cells usually remain, some form of therapy after complete remission is required to achieve long-term disease-free survival. Despite aggressive therapy, overall, only 20-30% of patients enjoy long-term disease-free survival.
In spite of advances that have occurred in the treatment of myelodysplastic syndrome and acute myeloid leukemia, it is clear that new approaches are needed to increase the fraction of patients cured.
II. Azacitidine and other DNA Methyltransferase Inhibitors
DNA Methyltransferase Inhibitors
DNA methylation is believed to play a key role in gene expression. DNA is methylated by DNA methyltransferases at the 5-position of the cytosine ring, almost exclusively in the context of CpG sites. CpG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. “CpG” stands for cytosine and guanine separated by a phosphate, which links the two nucleosides together in DNA. (The “CpG” notation is used to distinguish a cytosine followed by guanine from a cytosine base paired to a guanine.) The CpG sequence is relatively rare in eukaryotic genomes due to the action of DNA methyltransferases, which recognize these CpG sites and methylate the cytosine, turning it into 5-methylcytosine. Following spontaneous deamination, the 5-methylcytosine is converted into thymine.
However, there are regions of the DNA which have a high concentration of CpG sites. These regions, known as CpG islands, are found at the promoters of eukaryotic genes. These CpG sites are usually low in methylation. Methylation near the promoter sites inhibits gene expression.
DNA hypermethylation at CpG islands near the promoter region of genes is believed to be a key factor in diseases such as myelodysplastic syndrome, acute myeloid leukemia, and other malignancies because DNA methylation is a mechanism by which expression of genes can be inhibited. Thus, one approach to the treatment of such diseases has been the use of DNA methyltransferase inhibitors. Since DNA methylation is reversible, DNA methyltransferase inhibitors can be used to restore normal DNA methylation patterns, thereby reactivating genes involved in beneficial cellular functions such as controlling cellular proliferation, differentiation, apoptosis, and other homeostatic mechanism. Examples of such genes include cyclin dependent kinase 2a (p16), mutL homologue-1, and retinoblastoma. The scientific basis of this approach is discussed in detail by C. B. Yoo and P. A. Jones, Nature Rev., Drug Discovery, 2006, 5, 37-50, the entire disclosure of which is incorporated by reference.
There are four known DNA methyltransferase enzymes that have been characterized in detail, namely DNMT1, DNMT2, DNMT3a, and DNMT3b. In the DNA methyltransferases, the C-terminal catalytic domain is highly conserved. See B. Brueckner and F. Lyko, Trends Pharmacol. Sci., 2004, 25, 551-54.
There are two classes of DNA methyltransferase inhibitors, nucleoside analogues and non-nucleosides. The nucleoside analogues have a modified cytosine ring attached to either a ribose or deoxyribose moiety. Inhibition by such analogues is believed to occur when the nucleoside analogue is incorporated into DNA. Other DNA methyltransferase inhibitors are non-nucloside analogues. Examples of DNA methyltransferase inhibitors that are nucleoside analogues include azacitidine (5-azacytidine), decitabine (5-aza-2′-deoxycytidine), 5-fluoro-2′-deoxycitidine, 5,6-dihydro-5-azacytidine (DHAC), zebularine (2′-O-t-butyldimethylsilyl-3′-O-[(diisopropylamino)(2-cyanoethoxy)phosphino]-5′-O-(4,4′-dimethoxytrityl)-2(1H)-pyrimidinone-1-β-D-riboside), fazarabine (1-β-D-arabinofuranosyl-5-azacytosine). Among these, azacitidine and decitabine are believed to be particularly useful since they have shown clinical efficacy in the treatment of haemotological malignancies such as acute myeloid leukemia. Examples of non-nucleoside DNA methyltransferase inhibitors include hydralizine, procaine, procainamide, epigallocatechin gallate, psammaplin A, and RG108 ((S)-2-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-3-(1H-indol-3-yl)-propionic acid).
Azacitidine
Azacitidine is 5-azacytidine, a nucleoside analogue with the structure:

The compound has been studied in a number of clinical trials for the treatment of both solid tumors and leukemia. For a detailed discussion, see J. Goffin and E. Eisnhauer, Annals of Oncology, 2002, 13, 1699, the complete disclosure of which is incorporated by reference. Azacitidine is approved by the United States Food and Drug Administration as a drug for the treatment of myelodysplastic syndrome. In a phase 3 clinical trial of azacitidine given as a subcutaneous dose of 75 mg/m2/day for 7 days every 4 weeks to myelodysplastic syndrome patients, the response rate of 15% (5% of patients responded completely, and 10% of patients responded partially) was modest, albeit better than the complete lack of improvement seen in the control group. Details of the study are provided in the prescribing information for Vidaza™ (azacitidine) published by Pharmion Corporation, Aug. 31, 2004, the entire disclosure of which is incorporated by reference. Significant side-effects are seen with the drug, including nausea, anemia, thrombocytopenia, vomiting, pyrexia, leukopenia, diarrhea, fatigue, injection site erythema, constipation, neutropenia, and ecchymosis. In the clinical trials, leukopenia, thrombocytopenia and neutropenia were sufficiently serious to warrant reduction of the dose or discontinuation of treatment in some cases.
Because of a lack of available treatments for myelodysplastic syndrome and acute myeloid leukemia, and the toxicity and side effects associated with existing agents, the need exists for new therapies in the treatment of these diseases, particularly therapies that have greater potency and lower toxicity and/or activity across a broader spectrum of cell types. One solution would be a composition containing or method of using the above-mentioned therapeutic agents, wherein the efficacy is improved, for example by a synergistic combination with another compound. Such compositions or methods, could be very valuable in the treatment of myelodysplastic syndrome or acute myeloid leukemia. Using such compositions or methods in the treatment of myelodysplastic syndrome or acute myeloid leukemia could provide greater efficacy or potency, resulting in improved therapeutic response, diminished side effects, or both, as compared to using the above-mentioned chemotherapeutic agents alone.